Extraction and purification method of mouse sarcoma extracellular matrix

Abstract

The invention provides an extraction and purification method of a mouse sarcoma extracellular matrix. The method specifically comprises the following steps: (a) providing a sarcoma sample; (b) adding a NaCl impurity removal solution of which the volume is 1-3 times that of the sarcoma sample into the sarcoma sample, homogenizing, centrifuging, removing supernatant, and repeating the process of adding the NaCl impurity removal solution to remove impurities and supernatant so as to obtain a first precipitate containing sarcoma; (c) adding a urea extracting solution of which the volume is 1-2 times that of the first precipitate into the first precipitate, homogenizing and staying overnight to obtain a sarcoma overnight extracting solution; (d) centrifuging the overnight sarcoma extracting solution at a rotating speed of 32000-50000xg, taking a supernatant to obtain a second precipitate containing sarcoma, adding a urea extracting solution of which the volume is 0.3-0.8 time that of the second precipitate, homogenizing, and taking a supernatant; (e) combining the supernatant in the step (d), and uniformly mixing; and (f) dialyzing the supernatant in the step (e) to obtain the sarcoma extracellular matrix. According to the method disclosed by the invention, the mouse sarcoma extracellular matrix with high concentration, few impurities, stability and reliability can be obtained.

Description

Technical Field

[0001] The present invention relates to the biological field, and particularly to a method for extracting and purifying the extracellular matrix of mouse sarcoma cells. Background Art

[0002] The extracellular matrix (ECM) is a complex network composed of various macromolecules around cells in multicellular organisms. It is mainly composed of five types of substances: collagen, non-collagen, elastin, proteoglycan, and glycosaminoglycan. According to its distribution, it can be divided into two types: basement membrane and interstitial matrix. Among them, the interstitial matrix is mainly secreted by cells and is a loose collagen fiber network structure composed of components such as type I, type II, and type III collagen, fibronectin, elastin, and various proteoglycans; the basement membrane is a dense sheet-like protein network structure composed of type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan, which can separate cells from the surrounding matrix and play a barrier role in material transport. The extracellular matrix plays an important role in maintaining tissue structure, providing support and protecting cells. At the same time, it also plays an important role in many biological processes such as cell migration, cell adhesion, signal transduction, and tissue repair.

[0003] The extracellular matrix of mouse sarcoma cells, namely Matrigel, is a soluble basement membrane extract extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Its main components are laminin, type IV collagen, nidogen, heparan sulfate proteoglycan, and various growth factors. As a preparation extracted from the extracellular matrix, Matrigel retains the characteristics of the extracellular matrix and has the function of maintaining tissue shape, structure, and mechanical properties. At the same time, it can also provide structural support for the normal growth and physiological activities of cells, tissues, and organoids. Among them, various proteins and factors also play a coordinating and regulating role in cell growth. Therefore, it can be used as a biological scaffold material for three-dimensional cell culture to simulate the in vivo growth environment of cells and enable cells to better exert their functions and characteristics.

[0004] Matrigel derived from animals as a carrier for cell culture is closer to the in vivo growth environment of cells, can provide more accurate experimental data, and is widely used in medical tissue engineering, regenerative medicine, drug research and development, etc. It can be used as a biological scaffold for cell culture and tissue engineering to promote cell adhesion, proliferation, and differentiation. In addition, Matrigel can also be used for the construction of drug delivery systems to achieve precise drug release through drug loading and controlled release.

[0005] However, there are certain limitations in the source and preparation process of Matrigel, such as batch consistency, quality control, and standardization issues. In addition, the biocompatibility, stability, degradability, and biosafety of Matrigel also need to be further studied and optimized.

[0006] Therefore, in the application of Matrigel, continuous research needs to be carried out to solve these problems and improve the effectiveness and reliability of its application. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a method for extracting and purifying the extracellular matrix of mouse sarcoma cells. In the present invention, a tumor is inoculated into a normal mouse to obtain mouse sarcoma, and then it is extracted and purified with NaCl buffer solution and urea to obtain an extracellular matrix of mouse sarcoma with few impurities, stability and reliability. And further verified its good cell activity through 3D cell culture and cell proliferation experiments.

[0008] In the present invention, a method for purifying the extracellular matrix of sarcoma cells is provided, including the steps of:

[0009] (a) providing a sarcoma sample;

[0010] (b) adding 1-3 times (preferably 2 times) the volume of NaCl impurity removal solution to the sarcoma sample, homogenizing, centrifuging, removing the supernatant, and repeating the process of adding NaCl impurity removal solution to remove impurities and supernatant, so as to obtain a first precipitate containing sarcoma;

[0011] (c) adding 1-2 times the volume of urea extraction solution to the first precipitate, homogenizing, and incubating overnight to obtain a sarcoma overnight extraction solution;

[0012] (d) centrifuging the sarcoma overnight extraction solution at a rotation speed of 32000-50000 xg, taking the supernatant, obtaining a second precipitate containing sarcoma, adding 0.3-0.8 times (preferably 0.5 times) the volume of urea extraction solution to the second precipitate, homogenizing, and taking the supernatant;

[0013] (e) combining the supernatants in step (d), and mixing them evenly;

[0014] (f) dialyzing the supernatant in step (e) to obtain the extracellular matrix of sarcoma cells.

[0015] In another preferred example, the sarcoma sample is from a mouse.

[0016] In another preferred example, the NaCl impurity removal solution includes 100-250 g / L of NaCl, 1-5 g / L of Tris, 1-3 g / L of EDTA, and 1-4 g / L of NEM.

[0017] In another preferred example, the pH of the NaCl impurity removal solution is 7-8.

[0018] In another preferred example, the urea extraction solution includes 100 - 150 g / L of urea, 4 - 8 g / L of Tris, and 5 - 12 g / L of NaCl.

[0019] In another preferred example, in step (f), the dialysis time is greater than 2 h, preferably 3 - 5 h.

[0020] In another preferred example, in step (f), the number of dialysis times is 2.

[0021] In another preferred example, in step (d), the centrifugation time is 10 - 30 minutes.

[0022] In another preferred example, step (e) further includes step (e1): centrifuging again at a rotational speed of 32000 - 50000 xg to remove the precipitate.

[0023] In another preferred example, in step (e1), the centrifugation time is 40 - 80 minutes.

[0024] In another preferred example, the concentration of the sarcoma extracellular matrix is > 9 mg / ml, preferably 9 - 12 mg / ml.

[0025] In another preferred example, the method has one or more of the following characteristics selected from the group consisting of:

[0026] (a) The background impurities obtained are significantly reduced;

[0027] (b) The background of the sample is significantly removed, and there are almost no particulate impurities.

[0028] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. Description of the Drawings

[0029] Figure 1 It is a morphological diagram of the extracellular matrix obtained in Example 2 after solidification at 37°C.

[0030] Figure 2 It is a comparison diagram of the backgrounds of the extracellular matrices obtained in Examples 2 - 5.

[0031] Figure 3 It is the experimental result of the extracellular matrix prepared by the present invention for 3D cell culture.

[0032] Figure 4 It is the experimental result of the extracellular matrix prepared by the present invention for thin layer coating. Detailed Description of the Invention

[0033] After extensive and in-depth research and a large number of screenings, the inventor of the present invention has developed, for the first time, a method for extracting and purifying extracellular matrix of sarcoma cells. Specifically, in the method of the present invention, the present invention inoculates tumors into normal mice to obtain mouse sarcoma, and then extracts and purifies it with an NaCl impurity removal solution and a urea extraction solution, and centrifuges it at a specific rotational speed of 32000-50000 xg to obtain a mouse sarcoma extracellular matrix with high concentration, few impurities, and stability and reliability. On this basis, the present invention is completed.

[0034] The purification method of the present invention

[0035] The object of the present invention is to inoculate tumors into normal mice to obtain mouse sarcoma (which can be obtained by conventional methods or the method described in Example 1), and then extract and purify it with an NaCl buffer solution and a urea extraction solution to obtain a mouse sarcoma extracellular matrix with high concentration, few impurities, and stability and reliability. Furthermore, its good cell activity is verified through cell 3D culture and cell proliferation experiments.

[0036] Specifically, the present invention provides a method for purifying extracellular matrix of sarcoma cells, comprising the steps of:

[0037] (a) Providing a sarcoma sample;

[0038] (b) Adding 1-3 times (preferably 2 times) the volume of an NaCl impurity removal solution to the sarcoma sample, homogenizing, centrifuging, removing the supernatant, and repeating the process of adding the NaCl impurity removal solution to remove impurities and the supernatant to obtain a first precipitate containing sarcoma;

[0039] (c) Adding 1-2 times the volume of a urea extraction solution to the first precipitate, homogenizing, and incubating overnight to obtain an overnight extraction solution of sarcoma;

[0040] (d) Centrifuging the overnight extraction solution of sarcoma at a rotational speed of 32000-50000 xg, taking the supernatant to obtain a second precipitate containing sarcoma, adding 0.3-0.8 times (preferably 0.5 times) the volume of a urea extraction solution to the second precipitate, homogenizing, and taking the supernatant;

[0041] (e) Combining the supernatants in step (d), and mixing them evenly;

[0042] (f) Dialyzing the supernatant in step (e) to obtain extracellular matrix of sarcoma cells.

[0043] In a preferred embodiment, step (e1): centrifuging again at a rotational speed of 32000-50000 xg to remove the precipitate is further included in step (e).

[0044] In a preferred embodiment, in step (d), the centrifugation time is 10-30 minutes.

[0045] In a preferred embodiment, in step (e1), the centrifugation time is 50 - 80 minutes

[0046] The method of the present invention can obtain a mouse sarcoma extracellular matrix with high concentration, few impurities, and stable and reliable quality.

[0047] Application

[0048] The mouse sarcoma extracellular matrix with high concentration, few impurities, and stable and reliable quality obtained by the method of the present invention can be used in experiments such as invasion experiments, 3D culture, angiogenesis, and organoid culture.

[0049] The main advantages of the present invention include:

[0050] (1) For the first time, a method for extracting and purifying a sarcoma extracellular matrix is developed. Specifically, in the method of the present invention, the present invention inoculates a tumor into a normal mouse to obtain a mouse sarcoma, and then extracts and purifies it with an NaCl impurity removal solution and a urea extraction solution, and centrifuges it at a specific rotation speed of 32000 - 50000 xg, and a mouse sarcoma extracellular matrix with high concentration, few impurities, and stable and reliable quality can be obtained.

[0051] (2) The background impurities of the extracellular matrix obtained by the method of the present invention are significantly reduced. By extending the centrifugation time, the sample background removal is obvious, and there are almost no granular impurities.

[0052] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0053] Unless otherwise specified, the reagents and materials in the embodiments of the present invention are all commercially available products.

[0054] Example 1

[0055] (1) Turn on the ultraviolet disinfection of the laminar flow hood in advance for 30 min;

[0056] (2) Sacrifice the mouse by cervical dislocation;

[0057] (3) Spray the whole body of the mouse with 75% ethanol and then place the mouse on a pad. Take out the mouse sarcoma, take pictures, weigh, measure and record. If the dissected sarcoma contains a large amount of blood clots or a large amount of purulent substances, discard it.

[0058] (4) Take a certain amount of sarcoma mass, cut it into pieces with scissors, put it into a glass grinder. Take out the pre-cooled PBS, spray it with alcohol and put it into the laminar flow hood. Use a pipette to add a certain volume of pre-cooled PBS for grinding.

[0059] (5) Aspirate the ground mixture into a 50 mL centrifuge tube with a pipette, add a certain volume of pre-cooled PBS to each tube and shake well. After shaking, centrifuge at 1000 rpm for 5 - 10 min. If there are too many floating substances, it may be due to too high fat content and should be discarded.

[0060] (6) Prepare a 50 mL centrifuge tube to hold the waste liquid. Pour the supernatant into the centrifuge tube, then repeat the previous step, add PBS, shake well and centrifuge, and pour off the supernatant.

[0061] (7) Add a certain volume of double antibodies to the precipitate in the centrifuge tube (the volume of double antibodies accounts for 10% of the total volume), 20 mL of liquid in each tube (2 mL of double antibodies).

[0062] (8) Use a 1 mL syringe to inject evenly into the mice, 0.5 mL of sarcoma slurry for each mouse.

[0063] (9) After 3 - 4 weeks of growth of the inoculated mouse sarcoma for amplification, take out the mouse sarcoma, and the material collection method is the same as steps (1) - (3). Each 100 g is packed in a bag and stored in a -80 °C refrigerator.

[0064] Example 2

[0065] (1) Take out 100 g of mouse sarcoma from the -80 °C refrigerator and place it in a 4 °C refrigerator for overnight thawing.

[0066] (2) In the laminar flow hood, add TBS to the mouse sarcoma to wash away the blood in the sarcoma, repeat 2 times.

[0067] (3) Add 2 - fold volume of NaCl impurity removal solution (NaCl 198.5 g / L, Tris 3.03 g / L, EDTA 1.5 g / L, NEM 2.5 g / L, pH 7.4) to the mouse sarcoma, homogenize for 5 min with a homogenizer, then centrifuge at 8000 xg for 15 min at 4 °C to remove the supernatant. Repeat the NaCl impurity removal once.

[0068] (4) Add 1 - fold volume of urea extraction solution (urea 120 g / L, Tris 6.05 g / L, NaCl 9 g / L) to the precipitate, homogenize for 5 min, and extract overnight at 4 °C.

[0069] (5) The overnight sarcoma extract was centrifuged at 24,000 x g for 20 min at 4°C, the supernatant was taken, and 0.5 volume of the above urea extract was added to the sarcoma precipitate, homogenized again for 5 min, and centrifuged to take the supernatant.

[0070] (6) The supernatants were combined and mixed well.

[0071] (7) The extract was loaded into a 10KD dialysis bag and placed in TBS solution containing 1% chloroform, and dialyzed overnight.

[0072] (8) The dialysis bag was immersed in fresh TBS solution for 4 hours, and dialysis was repeated once with fresh TBS.

[0073] (9) The dialysis bag was immersed in high-glucose DMEM medium (purchased from Gibco, C11995500BT) and dialyzed overnight.

[0074] (10) In the ultra-clean bench on ice, the extracellular matrix was aliquoted into centrifuge tubes, and the protein concentration was measured by the BCA method to be 11.0 mg / mL. After the extracellular matrix solidified at 37°C as Figure 1 shown. The extracellular sarcoma samples were frozen in a -20°C refrigerator for later use.

[0075] Example 3

[0076] (1) 100 g of mouse sarcoma was taken out from an -80°C refrigerator and thawed overnight in a 4°C refrigerator.

[0077] (2) In the ultra-clean bench, TBS was added to the mouse sarcoma to wash away the blood in the sarcoma, and the operation was repeated 2 times.

[0078] (3) 2 volumes of NaCl impurity removal solution (NaCl 198.5 g / L, Tris 3.03 g / L, EDTA 1.5 g / L, NEM 2.5 g / L, pH 7.4) was added to the mouse sarcoma, homogenized with a homogenizer for 5 min, and then centrifuged at 8,000 x g for 15 min at 4°C to remove the supernatant. The NaCl impurity removal was repeated once.

[0079] (4) 1 volume of urea extract (urea 120 g / L, Tris 6.05 g / L, NaCl 9 g / L) was added to the precipitate, homogenized for 5 min, and extracted overnight at 4°C.

[0080] (5) The overnight sarcoma extract was centrifuged at 32,000 x g for 20 min at 4°C, the supernatant was taken, and 0.5 volume of the above urea extract was added to the sarcoma precipitate, homogenized again for 5 min, and centrifuged to take the supernatant.

[0081] (6) The supernatants were combined and mixed well.

[0082] (7) Load the extract into a 10KD dialysis bag and place it in a TBS solution containing 1% chloroform for overnight dialysis.

[0083] (8) Immerse the dialysis bag in fresh TBS solution for 4 hours of dialysis, and repeat the dialysis with fresh TBS once.

[0084] (9) Immerse the dialysis bag in DMEM high-glucose medium (purchased from Gibco, C11995500BT) for overnight dialysis.

[0085] (10) In the ultra-clean bench on ice, aliquot the extracellular matrix into centrifuge tubes. The protein concentration measured by the BCA method is 10.5 mg / mL. After the extracellular matrix solidifies at 37°C, as Figure 1 shown. The extracellular sarcoma samples are frozen in a -20°C refrigerator for later use.

[0086] Example 4

[0087] (1) Take out 100 g of mouse sarcoma from the -80°C refrigerator and place it in the 4°C refrigerator for overnight thawing.

[0088] (2) In the ultra-clean bench, add TBS to the mouse sarcoma to wash away the blood in the sarcoma, and repeat 2 times.

[0089] (3) Add 2 volumes of NaCl impurity removal solution (NaCl 198.5 g / L, Tris 3.03 g / L, EDTA 1.5 g / L, NEM 2.5 g / L, pH 7.4) to the mouse sarcoma, homogenize with a homogenizer for 5 min, and then centrifuge at 8000 xg for 15 min at 4°C to remove the supernatant. Repeat the NaCl impurity removal once.

[0090] (4) Add 1 volume of urea extraction solution (urea 120 g / L, Tris 6.05 g / L, NaCl 9 g / L) to the precipitate, homogenize for 5 min, and extract overnight at 4°C.

[0091] (5) Centrifuge the overnight sarcoma extract at 50000 xg for 20 min at 4°C, take the supernatant, and add 0.5 volume of the above urea extraction solution to the sarcoma precipitate, homogenize again for 5 min, and centrifuge to take the supernatant.

[0092] (6) Combine the supernatants and mix well.

[0093] (7) Load the extract into a 10KD dialysis bag and place it in a TBS solution containing 1% chloroform for overnight dialysis;

[0094] (8) Immerse the dialysis bag in fresh TBS solution for 4 hours of dialysis, and repeat the dialysis with fresh TBS once.

[0095] (9) Immerse the dialysis bag in DMEM high-glucose medium (purchased from Gibco, C11995500BT) and dialyze overnight.

[0096] (10) In the ultra-clean bench on ice, aliquot the extracellular matrix into centrifuge tubes. The protein concentration measured by the BCA method is 9.8 mg / mL. After the extracellular matrix solidifies at 37 °C, as Figure 1 shown. The extracellular sarcoma samples are frozen in a -20 °C refrigerator for later use.

[0097] Example 5

[0098] (1) Take out 100 g of mouse sarcoma from the -80 °C refrigerator and place it in the 4 °C refrigerator to thaw overnight.

[0099] (2) In the ultra-clean bench, add TBS to the mouse sarcoma to wash away the blood in the sarcoma, and repeat 2 times.

[0100] (3) Add 2 volumes of NaCl impurity removal solution (NaCl 198.5 g / L, Tris 3.03 g / L, EDTA 1.5 g / L, NEM 2.5 g / L, pH 7.4) to the mouse sarcoma, homogenize with a homogenizer for 5 min, then centrifuge at 8000 xg for 15 min at 4 °C to remove the supernatant. Repeat the NaCl impurity removal once.

[0101] (4) Add 1 volume of urea extraction solution (urea 120 g / L, Tris 6.05 g / L, NaCl 9 g / L) to the precipitate, homogenize for 5 min, and extract overnight at 4 °C.

[0102] (5) Centrifuge the overnight sarcoma extract at 32000 xg for 20 min at 4 °C, take the supernatant, and add 0.5 volume of the above urea extraction solution to the sarcoma precipitate, homogenize again for 5 min, and centrifuge to take the supernatant.

[0103] (6) Combine the supernatants and mix well. Centrifuge again at 32000 xg for 60 min at 4 °C to remove the precipitate.

[0104] (7) Load the extract into a 10KD dialysis bag and place it in a TBS solution containing 1% chloroform, and dialyze overnight.

[0105] (8) Immerse the dialysis bag in fresh TBS solution for 4 hours, and repeat dialysis with fresh TBS once.

[0106] (9) Immerse the dialysis bag in DMEM high-glucose medium (purchased from Gibco, C11995500BT) and dialyze overnight.

[0107] (10) In a clean bench, on ice, the extracellular matrix was dispensed into centrifuge tubes. The protein concentration was measured by BCA method and was 9.3 mg / mL. The extracellular sarcoma sample was frozen in a -20°C refrigerator for later use.

[0108] Example 6

[0109] (1) Place the matrix gel on ice and thaw at 2-8°C overnight.

[0110] (2) Place an ice box (with crushed ice) in a biosafety cabinet and irradiate with UV light for 30 min. Place the reagents in a water bath for 30 min.

[0111] (3) HepG2 cells (purchased from Pronocell) were taken out from the CO2 incubator, and the cell status and confluence were observed under a microscope. If the status was normal, they were placed in a biosafety cabinet.

[0112] (4) Discard the supernatant, add PBS buffer, rinse the cells, and discard the supernatant.

[0113] (5) Add 0.25% trypsin solution to wet the cells, then remove the trypsin solution and place the cells in a 37°C carbon dioxide incubator for 4-5 minutes. Observe the cell digestion status under a microscope.

[0114] (6) After digestion is complete, add complete medium to terminate digestion, mix thoroughly by pipetting, and take 200 μL for counting. (7) Pre-cool a 1.5 mL centrifuge tube on ice and adjust the cell suspension density to 5 × 10 5 cells / mL, take cell suspension: matrix gel = 1:3, and mix gently by pipetting (avoiding bubbles).

[0115] (8) Add 20 μL of the mixed solution (i.e., the mixed solution of cell suspension in step (7) and matrix gel in a ratio of 1:3) to each well, drop it in the 24-well plate, gently flip the plate over so that the droplet forms an arch, let it stand at room temperature for 30 seconds, and then operate the subsequent samples in sequence. After completion, invert the plate and place it in a 37°C incubator for incubation for 30 minutes.

[0116] (9) After complete gelation, add 1 mL of complete culture medium (purchased from Gibco) to each well.

[0117] (10) Take photos and record them at 0d, 3d, 5d, 7d, and 10d. Add 0.5 mL of complete culture medium after taking photos at 3d, 5d, and 7d.

[0118] Example 7

[0119] (1) The experiment set up a test sample group, a negative control group and a positive control group.

[0120] Test sample group: NIH-3T3 cells (purchased from Procell) were cultured in a T75 culture flask coated with a thin layer of Matrigel.

[0121] Positive control group: NIH-3T3 cells were cultured in a T75 culture flask coated with a thin layer of CORNING Matrigel.

[0122] Negative control group: NIH-3T3 cells were cultured in a T75 culture flask not coated with a thin layer of Matrigel.

[0123] (2) Sample treatment: Dilute the Matrigel sample to the corresponding working solution concentration of 50 - 200 μg / mL.

[0124] (3) Thin layer coating: The coating concentration is 5 - 20 μg / cm 2 Add the working solutions of the test sample group and the positive control group to the culture flask, shake well to evenly cover the culture surface with the sample, place the culture flask in an incubator at 37°C for incubation, and aspirate and discard the coating solution after incubation.

[0125] (4) Seeding: Add cell suspension to the test sample group, negative control group, and positive control group, slowly add it along the wall of the flask to the culture flask, shake well, and place it in a 37°C, 5% carbon dioxide incubator for culture.

[0126] (5) After culturing for a period of time, place it under an inverted microscope for observation and photography, and compare the cell growth confluence and cell morphology in each flask.

[0127] Experimental results:

[0128] Figure 1 It is the morphological diagram of the extracellular matrix obtained in Example 2 after solidification at 37°C; the extracellular matrix rapidly forms a gel at 37°C.

[0129] Figure 2 It is the background comparison diagram of the extracellular matrix in Examples 2 - 5. After the extraction centrifugation speed is increased from 24000 xg to 32000 xg, the background impurities are significantly reduced; when the centrifugation speed is further increased to 50000 xg, the result is similar to that under the condition of 32000 xg; in Example 5, the extracted supernatant is centrifuged again and the centrifugation time is extended, and the sample background is significantly removed and there are almost no particulate impurities.

[0130] Figure 3 It is the comparison result of the extracellular matrix prepared by the present invention in Example 6 and the extracellular matrix of Corning used in 3D cell culture experiments; HepG2 cells are cultured in 3D spheroids for 10 days, and the cell viability is consistent with that of Corning.

[0131] Figure 4Comparison results of the extracellular matrix prepared in Example 7 of the present invention and the extracellular matrix of Corning Incorporated for thin-layer coating experiments; the thin-layer coating effect of the sample prepared in the present invention is good, and there is no significant difference in the ability to promote cell proliferation compared with Corning (2174002), and both are significantly better than the negative control.

[0132] The embodiments of the present invention have been further described above in conjunction with the accompanying drawings and specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can be made, and all should be regarded as belonging to the protection scope of the present invention.

[0133] All the documents mentioned in the present invention are cited herein as references, just as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for purifying a sarcoma extracellular matrix, characterized in that, Including the steps: (a) Providing a sarcoma sample; (b) Adding a NaCl impurity removal solution with a volume 1 - 3 times (preferably 2 times) that of the sarcoma sample, homogenizing, centrifuging, removing the supernatant, and repeating the process of adding the NaCl impurity removal solution for impurity removal and removing the supernatant to obtain a first precipitate containing sarcoma; (c) Adding a urea extraction solution with a volume 1 - 2 times that of the first precipitate, homogenizing, and incubating overnight to obtain an overnight sarcoma extraction solution; (d) Centrifuging the overnight sarcoma extraction solution at a rotational speed of 32000 - 50000 xg, taking the supernatant, obtaining a second precipitate containing sarcoma, adding a urea extraction solution with a volume 0.3 - 0.8 times (preferably 0.5 times) that of the second precipitate, homogenizing, and taking the supernatant; (e) Combining the supernatants in step (d) and mixing well; (f) Dialyzing the supernatant in step (e) to obtain an extracellular matrix of sarcoma cells.

2. The method according to claim 1, wherein The NaCl impurity removal solution includes 100 - 250 g / L of NaCl, 1 - 5 g / L of Tris, 1 - 3 g / L of EDTA, and 1 - 4 g / L of NEM.

3. The method according to claim 1, characterized in that The pH of the NaCl impurity removal solution is 7 - 8.

4. The method according to claim 1, wherein The urea extraction solution includes 100 - 150 g / L of urea, 4 - 8 g / L of Tris, and 5 - 12 g / L of NaCl.

5. The method according to claim 1, wherein In step (f), the dialysis time is greater than 2 h, preferably 3 - 5 h.

6. The method according to claim 1, wherein In step (f), the number of dialysis times is 2 times.

7. The method according to claim 1, characterized in that In step (d), the centrifugation time is 10 - 30 minutes.

8. The method according to claim 1, wherein Step (e) also includes step (e1): centrifuging again at a rotational speed of 32000 - 50000 xg to remove the precipitate.

9. The method according to claim 8, wherein In step (e1), the centrifugation time is 40 - 80 minutes.

10. The method according to claim 1, wherein The concentration of the extracellular matrix of sarcoma cells is > 9 mg / ml, preferably 9 - 12 mg / ml.

Images